Humanized anti-factor d antibodies and uses thereof

ABSTRACT

The invention relates to humanized anti-human Factor D monoclonal antibodies, their nucleic acid and amino acid sequences, the cells and vectors that harbor these antibodies and their use in the preparation of compositions and medicaments for treatment of diseases and disorders associated with excessive or uncontrolled complement activation. These antibodies are useful for diagnostics, prophylaxis and treatment of disease.

BACKGROUND OF THE INVENTION

The complement system plays a central role in the clearance of immunecomplexes and the immune response to infectious agents, foreignantigens, virus-infected cells and tumor cells. However, complement isalso involved in pathological inflammation and in autoimmune diseases.Therefore, inhibition of excessive or uncontrolled activation of thecomplement cascade could provide clinical benefit to patients with suchdiseases and conditions.

The complement system encompasses two distinct activation pathways,designated the classical and the alternative pathways (V. M. Holers, InClinical Immunology: Principles and Practice, ed. R. R. Rich, MosbyPress; 1996, 363-391). The classical pathway is acalcium/magnesium-dependent cascade which is normally activated by theformation of antigen-antibody complexes. The alternative pathway is amagnesium-dependent cascade which is activated by deposition andactivation of C3 on certain susceptible surfaces (e.g. cell wallpolysaccharides of yeast and bacteria, and certain biopolymermaterials). Activation of the complement pathway generates biologicallyactive fragments of complement proteins, e.g. C3a, C4a and C5aanaphylatoxins and C5b-9 membrane attack complexes (MAC), which mediateinflammatory activities involving leukocyte chemotaxis, activation ofmacrophages, neutrophils, platelets, mast cells and endothelial cells,vascular permeability, cytolysis, and tissue injury.

Factor D is a highly specific serine protease essential for activationof the alternative complement pathway. It cleaves factor B bound to C3b,generating the C3b/Bb enzyme which is the active component of thealternative pathway C3/C5 convertases. Factor D may be a suitable targetfor inhibition, since its plasma concentration in humans is very low(1.8 μg/ml), and it has been shown to be the limiting enzyme foractivation of the alternative complement pathway (P. H. Lesavre and H.J. Müller-Eberhard. J. Exp. Med., 1978; 148: 1498-1510; J. E. Volanakiset al., New Eng. J. Med., 1985; 312: 395-401).

The down-regulation of complement activation has been demonstrated to beeffective in treating several disease indications in animal models andin ex vivo studies, e.g. systemic lupus erythematosus andglomerulonephritis (Y. Wang et al., Proc. Natl. Acad. Sci.; 1996, 93:8563-8568), rheumatoid arthritis (Y. Wang et al., Proc. Natl. Acad.Sci., 1995; 92: 8955-8959), cardiopulmonary bypass and hemodialysis (C.S. Rinder, J. Clin. Invest., 1995; 96: 1564-1572), hypercute rejectionin organ transplantation (T. J. Kroshus et al., Transplantation, 1995;60: 1194-1202), myocardial infarction (J. W. Homeister et al., J.Immunol., 1993; 150: 1055-1064; H. F. Weisman et al., Science, 1990;249: 146-151), reperfusion injury (E. A. Amsterdam et al., Am. J.Physiol., 1995; 268: H448-H457), and adult respiratory distress syndrome(R. Rabinovici et al., J. Immunol., 1992; 149: 1744-1750). In addition,other inflammatory conditions and autoimmune/immune complex diseases arealso closely associated with complement activation (V. M. Holers, ibid.,B. P. Morgan. Eur. J. Clin. Invest., 1994: 24: 219-228), includingthermal injury, severe asthma, anaphylactic shock, bowel inflammation,urticaria, angioedema, vasculitis, multiple sclerosis, myastheniagravis, membranoproliferative glomerulonephritis, and Sjögren'ssyndrome.

There is a need for antibody therapeutics in the field ofcomplement-mediated disorders and the humanized antibodies of thepresent invention provide high affinity antibodies useful to meet thisneed.

SUMMARY OF THE INVENTION

The present invention relates generally to antibodies comprising theheavy and light chain variable domain sequences of murine antibody166-32, which is an antibody capable of inhibiting biological activitiesassociated with Factor D. For example, at a concentration of 18 μg/ml(equivalent to about 1.5 times the molar concentration of human factor Din the blood; molar ratio of anti-Factor D antibody to Factor D of about1.5:1), significant inhibition of the alternative complement activity bythe antibody can be observed (see, e.g., U.S. Pat. No. 6,956,107)

The present invention also relates to humanized antibodies of murine MAb166-32. The invention includes the amino acid sequences of the variableheavy and light chain of the antibodies and their corresponding nucleicacid sequences. Another embodiment of the invention includes the CDRsequences of these antibodies.

Another embodiment of the present invention includes compositionscomprising an antibody of the invention. In another embodiment, theinvention provides cell lines and vectors harboring the antibodysequences of the present invention. In one aspect, the inventionincludes method of making and using antibodies and compositions of theinvention.

Another embodiment of the preset invention is the use of these humanizedantibodies for the preparation of a medicament or composition for thetreatment of disorders associated with excessive or uncontrolledcomplement activation. They include complement activation duringcardiopulmonary bypass operations; complement activation due toischemia-reperfusion following acute myocardial infarction, aneurysm,stroke, hemorrhagic shock, crush injury, multiple organ failure,hypobolemic shock, intestinal ischemia or other events causing ischemia.Complement activation has also been shown to be associated withinflammatory conditions such as severe burns, endotoxemia, septic shock,adult respiratory distress syndrome, hemodialysis; anaphylactic shock,severe asthma, angioedema, Crohn's disease, sickle cell anemia,poststreptococcal glomerulonephritis and pancreatitis. The disorder maybe the result of an adverse drug reaction, drug allergy, IL-2 inducedvascular leakage syndrome or radiographic contrast media allergy. Italso includes autoimmune disease such as systemic lupus erythematosus,myasthenia gravis, rheumatoid arthritis, Alzheimer's disease andmultiple sclerosis. Complement activation is also associated withtransplant rejection. Complement activation is also associated withocular diseases such as age-related macular degeneration, diabeticretinopathy.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B depict the amino acid sequence of the Murine MAb 166-32Variable Heavy Chain (FIG. 1A) and the Variable Light Chain (FIG. 1B).

FIGS. 2A and 2B depict nucleic acid sequence of the Murine MAb 166-32Variable Heavy Chain (FIG. 2A) and the Variable Light Chain (FIG. 2B).

FIG. 3 depicts the comparison of the light chains of the murine MAb166-32.

FIG. 4 depicts the comparison of the heavy chains of the murine MAb166-32.

FIG. 5 depicts the amino acid sequences of the Variable Heavy Chain andthe Variable Light Chain for each humanized antibody clone #56, #111,#250, and #416.

FIG. 6 depicts the hemolytic assay results for humanized antibody Fabclone #56, #111, #250, and #416.

FIG. 7 depicts the inhibition of the alternative complement activity byhumanized antibody Fab clones #56, #111, #250, and #416.

FIG. 8A-B (variable heavy (VH) consensus frameworks) and

FIG. 9A-B (variable light (VL) consensus frameworks) depict exemplaryacceptor human consensus framework sequences that can be used inpracticing the instant invention with sequence identifiers as follows:(FIG. 8A-B) human VH subgroup I consensus framework minus Kabat CDRs(SEQ ID NO: 28), human VH subgroup I consensus framework minus extendedhypervariable regions (SEQ ID NOs: 29-31), human VH subgroup IIconsensus framework minus Kabat CDRs (SEQ ID NO: 32), human VH subgroupII consensus framework minus extended hypervariable regions (SEQ ID NOs:33-35), human VH subgroup III consensus framework minus Kabat CDRs (SEQID NO: 36), human VH subgroup III consensus framework minus extendedhypervariable regions (SEQ ID NOs: 37-39), human VH subgroup VIIconsensus framework minus Kabat CDRs (SEQ ID NO: 55), human VH subgroupVII consensus framework minus extended hypervariable regions (SEQ IDNOs: 56-58), human VH acceptor framework minus Kabat CDRs (SEQ ID NO:40), human VH acceptor framework minus extended hypervariable regions(SEQ ID NOs: 41-42), human VH acceptor 2 framework minus Kabat CDRs (SEQID NO: 43) and human VH acceptor 2 framework minus extendedhypervariable regions (SEQ ID NOs: 44-46) and (FIG. 9A-B) human VL kappasubgroup I consensus framework (SEQ ID NO: 47), human VL kappa subgroupconsensus framework (SEQ ID NO: 48), human kappa subgroup III consensusframework (SEQ ID NO: 49) and human kappa subgroup IV consensusframework (SEQ ID NO: 50).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms used throughout this application are to be construed with ordinaryand typical meaning to those of ordinary skill in the art. However,Applicants desire that the following terms be given the particulardefinition as defined below.

The phrase “substantially identical” with respect to an antibody chainpolypeptide sequence may be construed as an antibody chain exhibiting atleast 70%, or 80%, or 90% or 95% sequence identity to the referencepolypeptide sequence. The term with respect to a nucleic acid sequencemay be construed as a sequence of nucleotides exhibiting at least about85%, or 90%, or 95% or 97% sequence identity to the reference nucleicacid sequence.

The term “identity” or “homology” shall be construed to mean thepercentage of amino acid residues in the candidate sequence that areidentical with the residue of a corresponding sequence to which it iscompared, after aligning the sequences and introducing gaps, ifnecessary to achieve the maximum percent identity for the entiresequence, and not considering any conservative substitutions as part ofthe sequence identity. Neither N- or C-terminal extensions norinsertions shall be construed as reducing identity or homology. Methodsand computer programs for the alignment are well known in the art.Sequence identity may be measured using sequence analysis software.

The term “antibody” is used in the broadest sense, and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, and multispecific antibodies (e.g.,bispecific antibodies). Antibodies (Abs) and immunoglobulins (Igs) areglycoproteins having the same structural characteristics. Whileantibodies exhibit binding specificity to a specific target,immunoglobulins include both antibodies and other antibody-likemolecules which lack target specificity. Native antibodies andimmunoglobulins are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each heavy chain has at one end a variabledomain (V_(H)) followed by a number of constant domains. Each lightchain has a variable domain at one end (V_(L)) and a constant domain atits other end.

As used herein, “anti-human Factor D antibody” means an antibody whichspecifically binds to human Factor D in such a manner so as to inhibitor substantially reduce complement activation.

The term “variable” in the context of variable domain of antibodies,refers to the fact that certain portions of the variable domains differextensively in sequence among antibodies and are used in the binding andspecificity of each particular antibody for its particular target.However, the variability is not evenly distributed through the variabledomains of antibodies. It is concentrated in three segments calledcomplementarity determining regions (CDRs) also known as hypervariableregions (HVRs) both in the light chain and the heavy chain variabledomains. The more highly conserved portions of variable domains arecalled the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely a adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of the targetbinding site of antibodies (see Kabat et al.). As used herein, numberingof immunoglobulin amino acid residues is done according to theimmunoglobulin amino acid residue numbering system of Kabat et al.,(Sequences of Proteins of Immunological Interest, National Institute ofHealth, Bethesda, Md. 1987), unless otherwise indicated.

The term “hypervariable region”, “HVR”, or “HV”, when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH (H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions representa compromise between the Kabat CDRs and Chothia structural loops, andare used by Oxford Molecular's AbM antibody modeling software. The“contact” hypervariable regions are based on an analysis of theavailable complex crystal structures. The residues from each of thesehypervariable regions are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in theVL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3)in the VH. The variable domain residues are numbered according to Kabatet al., supra for each of these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues or CDR residues herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat”, and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g, Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra; hinge region in constant domain of heavy chainis approximately residues 216-230 (EU numbering) of the heavy chain).The “EU index as in Kabat” refers to the residue numbering of the humanIgG1 EU antibody. Unless stated otherwise herein, references to residuenumbers in the variable domain of antibodies means residue numbering bythe Kabat numbering system. Unless stated otherwise herein, referencesto residue numbers in the constant domain of antibodies means residuenumbering by the EU numbering system (e.g., see U.S. ProvisionalApplication No. 60/640,323, Figures for EU numbering).

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the target binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂ and Fv fragments. Thephrase “functional fragment or analog” of an antibody is a compoundhaving qualitative biological activity in common with a full-lengthantibody. For example, a functional fragment or analog of an anti-humanFactor D antibody is one which can bind to Factor D in such a manner soas to prevent or substantially reduce the complement activation. As usedherein, “functional fragment” with respect to antibodies, refers to Fv,F(ab) and F(ab′)₂ fragments. An “Fv” fragment is the minimum antibodyfragment which contains a complete target recognition and binding site.This region consists of a dimer of one heavy and one light chainvariable domain in a tight, non-covalent association (V_(H)-V_(L)dimer). It is in this configuration that the three CDRs of each variabledomain interact to define an target binding site on the surface of theV_(H)-V_(L) dimer. Collectively, the six CDRs confer target bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three CDRs specific for an target) has theability to recognize and bind target. “Single-chain Fv” or “sFv”antibody fragments comprise the V_(H) and V_(L) domains of an antibody,wherein these domains are present in a single polypeptide chain.Generally, the Fv polypeptide further comprises a polypeptide linkerbetween the V_(H) and V_(L) domains which enables the sFv to form thedesired structure for target binding.

The Fab fragment contains the constant domain of the light chain and thefirst constant domain (CH1) of the heavy chain. Fab′ fragments differfrom Fab fragments by the addition of a few residues at the carboxylterminus of the heavy chain CH1 domain including one or more cysteinesfrom the antibody hinge region. F(ab′) fragments are produced bycleavage of the disuffide bond at the hinge cysteines of the F(ab′)₂pepsin digestion product. Additional chemical couplings of antibodyfragments are known to those of ordinary skill in the art.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single targetic site. Furthermore, in contrast to conventional(polyclonal) antibody preparations which typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on thetarget. In addition to their specificity, monoclonal antibodies areadvantageous in that they may be synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monodonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies for use with the presentinvention may be isolated from phage antibody libraries using the wellknown techniques. The parent monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler and Milstein, Nature 256, 495 (1975),or may be made by recombinant methods.

“Humanized” forms of non-human (e.g. murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other target-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin consensus sequence. The humanized antibody mayalso comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin template chosen.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies can in some instances be important toreduce antigenicity and/or HAMA response (human anti-mouse antibody)when the antibody is intended for human therapeutic use. Reduction orelimination of a HAMA response is generally a significant aspect ofclinical development of suitable therapeutic agents. See, e.g.,Khaxzaeli et al., J. Natl. Cancer Inst. (1988), 80:937; Jaffers et al.,Transplantation (1986), 41:572; Shawler et al., J. Immunol. (1985),135:1530; Sears et al., J. Biol. Response Mod. (1984), 3:138; Miller etal., Blood (1983), 62:988; Hakimi et al., J. Immunol. (1991), 147:1352;Reichmann et al., Nature (1988), 332:323; Junghans et al., Cancer Res.(1990), 50:1495. As described herein, the invention provides antibodiesthat are humanized such that HAMA response is reduced or eliminated.Variants of these antibodies can further be obtained using routinemethods known in the art, some of which are further described below.According to the so-called “best-fit” method, the sequence of thevariable domain of a rodent antibody is screened against the entirelibrary of known human variable domain sequences. The human V domainsequence which is closest to that of the rodent is identified and thehuman framework region (FR) within it accepted for the humanizedantibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J.Mol. Biol., 196:901 (1987)). Another method uses a particular frameworkregion derived from the consensus sequence of all human antibodies of aparticular subgroup of light or heavy chains. The same framework may beused for several different humanized antibodies (Carter et al., Proc.Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol.151:2623 (1993)).

For example, an amino acid sequence from an antibody as described hereincan serve as a starting (parent) sequence for diversification of theframework and/or hypervariable sequence(s). A selected frameworksequence to which a starting hypervariable sequence is linked isreferred to herein as an acceptor human framework. While the acceptorhuman frameworks may be from, or derived from, a human immunoglobulin(the VL and/or VH regions thereof), the acceptor human frameworks may befrom, or derived from, a human consensus framework sequence as suchframeworks have been demonstrated to have minimal, or no, immunogenicityin human patients. An “acceptor human framework” for the purposes hereinis a framework comprising the amino acid sequence of a VL or VHframework derived from a human immunoglobulin framework, or from a humanconsensus framework. An acceptor human framework “derived from” a humanimmunoglobulin framework or human consensus framework may comprise thesame amino acid sequence thereof, or may contain pre-existing amino acidsequence changes. Where pre-existing amino acid changes are present,preferably no more than 5 and preferably 4 or less, or 3 or less,pre-existing amino acid changes are present. In one embodiment, the VHacceptor human framework is identical in sequence to the VH humanimmunoglobulin framework sequence or human consensus framework sequence.In one embodiment, the VL acceptor human framework is identical insequence to the VL human immunoglobulin framework sequence or humanconsensus framework sequence. A “human consensus framework” is aframework which represents the most commonly occurring amino acidresidue in a selection of human immunoglobulin VL or VH frameworksequences. Generally, the selection of human immunoglobulin VL or VHsequences is from a subgroup of variable domain sequences. Generally,the subgroup of sequences is a subgroup as in Kabat et al. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal. In one embodiment, for the VH, the subgroup is subgroup III as inKabat et al.

Where the acceptor is derived from a human immunoglobulin, one mayoptionally select a human framework sequence that is selected based onits homology to the donor framework sequence by aligning the donorframework sequence with various human framework sequences in acollection of human framework sequences, and select the most homologousframework sequence as the acceptor. The acceptor human framework may befrom or derived from human antibody germline sequences available in thepublic databases.

In one embodiment, human consensus frameworks herein are from, orderived from, VH subgroup VII and/or VL kappa subgroup I consensusframework sequences.

In one embodiment, the human framework template used for generation ofan anti-Factor D antibody may comprise framework sequences from atemplate comprising a combination of VI-4.1 b+ (VH7 family) and JH4d forVH chain (FIG. 3) and/or a combination of DPK4 (VκI family) and JK2 forVL chain (FIG. 4).

Thus, the VH acceptor human framework may comprise one, two, three orall of the following framework sequences: FR1 comprisingQX₁QLVQSGX₂ELKKPGASVKVSCKAS (amino acids 1-25 of SEQ ID NO: 27), whereinX₁ is I or V, X₂ is P or S; FR2 comprising WVX₃QAPGQGLE (amino acids36-46 of SEQ ID NO: 27), wherein X₃ is K or R; FR3 comprisingRFVFSLDTSVSTAYLQISSLKAEDTAX₄YYCX₅R (amino acids 67-98 of SEQ ID NO: 27),wherein X₄ is T or V, X₅ is E or A; FR4 comprising WGQGTLVTVSS (aminoacids 105-115 of SEQ ID NO: 8 or amino acids 105-115 of SEQ ID NO: 27)

Examples of VH consensus frameworks include:

human VH subgroup I consensus framework minus Kabat CDRs (SEQ ID NO:28);human VH subgroup I consensus framework minus extended hypervariableregions (SEQ ID NOs: 29-31);human VH subgroup II consensus framework minus Kabat CDRs (SEQ ID NO:32);human VH subgroup II consensus framework minus extended hypervariableregions (SEQ ID NOs: 33-35);human VH subgroup III consensus framework minus Kabat CDRs (SEQ ID NO:36);human VH subgroup III consensus framework minus extended hypervariableregions (SEQ ID NO: 37-39);human VH subgroup VII consensus framework minus Kabat CDRs (SEQ ID NO:55);human VH subgroup VII consensus framework minus extended hypervariableregions (SEQ ID NO: 56-58);human VH acceptor framework minus Kabat CDRs (SEQ ID NO: 40);human VH acceptor framework minus extended hypervariable regions (SEQ IDNOs: 41-42);human VH acceptor 2 framework minus Kabat CDRs (SEQ ID NO: 43); orhuman VH acceptor 2 framework minus extended hypervariable regions (SEQID NOs: 44-45).

In one embodiment, the VH acceptor human framework comprises one, two,three or all of the following framework sequences:

FR1 comprising QVQLVQSGPELKKPGASVKVSCKAS (amino acids 1-25 of SEQ ID NO:8),FR2 comprising WVRQAPGQGLE (amino acids 36-46 of SEQ ID NO: 8),FR3 comprising RFVFSLDTSVSTAYLQISSLKAEDTAVYYCER (amino acids 67-98 ofSEQ ID NO: 8),RFVFSLDTSVSTAYLQISSLKAEDTAVYYCE (amino acids 67-97 of SEQ ID NO: 8),RFVFSLDTSVSTAYLQISSLKAEDTAVYYC (amino acids 67-96 of SEQ ID NO: 8),

RFVFSLDTSVSTAYLQISSLKAEDTAVYYCS (SEQ ID NO: 51), orRFVFSLDTSVSTAYLQISSLKAEDTAVYYCSR (SEQ ID NO: 52)

FR4 comprising WGQGTLVTVSS (amino acids 105-115 of SEQ ID NO: 8 or aminoacids 105-115 of SEQ ID NO: 27).

The VL acceptor human framework may comprise one, two, three or all ofthe following framework sequences:

FR1 comprising DIQX₆TQSPSSLSX₇SVGDRVTITC (amino acids 1-23 of SEQ ID NO:26), wherein X₆ is V or M, X₇ is M or A;FR2 comprising WYQQKPGKX₈PKLLIX₉ (amino acids 35-49 of SEQ ID NO: 26),wherein X₈ is P or V, X₉ is S or Y;FR3 comprising GVPSRFSX₁₀SGSGX₁₁DFTLTISSLQPEDVATYYC (amino acids 57-88of SEQ ID NO: 26), wherein X₁₀ is S or G, X₁₁ is A or T;FR4 comprising FGQGTKX₁₂EIK (SEQ ID NO: 54), wherein X₁₂ is V or L.

Examples of VL consensus frameworks include:

human VL kappa subgroup I consensus framework (SEQ ID NO: 47);human VL kappa subgroup II consensus framework (SEQ ID NO: 48);human VL kappa subgroup III consensus framework (SEQ ID NO: 49); orhuman VL kappa subgroup IV consensus framework (SEQ ID NO: 50)

In one embodiment, the VL acceptor human framework may comprise one,two, three or all of the following framework sequences:

FR1 comprising DIQVTQSPSSLSASVGDRVTITC (amino acids 1-23 of SEQ ID NO:7),FR2 comprising WYQQKPGKVPKLLIS (amino acids 35-49 of SEQ ID NO: 7),FR3 comprising GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (amino acids 57-88 ofSEQ ID NO: 7),FR4 comprising FGQGTKLEIK (amino acids 98-107 of SEQ ID NO: 7), orFGQGTKVEIK (SEQ ID NO: 53).

While the acceptor may be identical in sequence to the human frameworksequence selected, whether that be from a human immunoglobulin or ahuman consensus framework, the present invention contemplates that theacceptor sequence may comprise pre-existing amino acid substitutionsrelative to the human immunoglobulin sequence or human consensusframework sequence. These pre-existing substitutions are preferablyminimal; usually four, three, two or one amino acid differences onlyrelative to the human immunoglobulin sequence or consensus frameworksequence.

Hypervariable region residues of the non-human antibody are incorporatedinto the VL and/or VH acceptor human frameworks. For example, one mayincorporate residues corresponding to the Kabat CDR residues, theChothia hypervariable loop residues, the Abm residues, and/or contactresidues. Optionally, the extended hypervariable region residues asfollows are incorporated: 24-34 (L1), 50-56 (L2) and 89-97 (L3), 26-35(H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3).

In one aspect, the invention provides an antibody comprising at leastone, two, three, four, five or six HVRs selected from (a) an HVR-H1comprising the amino acid sequence selected from SEQ ID NO: 13 and SEQID NO: 25; (b) an HVR-H2 comprising the amino acid sequence of SEQ IDNO: 14; (c) an HVR-H3 comprising the amino acid sequence selected fromSEQ ID NO: 15 and SEQ ID NO: 20; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 16; (e) an HVR-L2 comprising the amino acidsequence selected from SEQ ID NO: 17, SEQ ID NO; 21 and SEQ ID NO: 23;and (f) an HVR-L3 comprising the amino acid sequence selected from SEQID NO: 18, SEQ ID NO: 22 and SEQ ID NO: 24.

In one aspect, the invention provides an anti-Factor D antibodycomprising at least one, two, three, four, five or six HVRs selectedfrom (a) an HVR-H1 comprising an amino acid sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity toan amino acid sequence selected from SEQ ID NO: 13 and SEQ ID NO: 25;(b) an HVR-H2 comprising an amino acid sequence having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to anamino acid sequence of SEQ ID NO: 14; (c) an HVR-H3 comprising an aminoacid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% sequence identity to an amino acid sequence selected from SEQID NO: 15 and SEQ ID NO: 20; (d) an HVR-L1 comprising an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to an amino acid sequence of SEQ ID NO: 16; (e) anHVR-L2 comprising an amino acid sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acidsequence selected from SEQ ID NO: 17, SEQ ID NO; 21 and SEQ ID NO: 23;and (f) an HVR-L3 comprising an amino acid sequence having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to anamino acid sequence selected from SEQ ID NO: 18, SEQ ID NO: 22 and SEQID NO: 24. In some embodiments, an HVR having an amino acid sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity contains substitutions, insertions, or deletionsrelative to the reference sequence, but an antibody comprising thatamino acid sequence retains the ability to bind to Factor D. In someembodiments, a total of 1 to 10 amino acids have been substituted,inserted, or deleted in the reference sequence selected from the groupconsisting of SEQ ID NO: 13, SEQ ID NO: 25, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 20, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO; 21, SEQ IDNO: 23, SEQ ID NO: 18, SEQ ID NO: 22 and SEQ ID NO: 24. In someembodiments, the invention provides an antibody comprising at least one,two, three, four, five or six HVRs selected from (a) an HVR-H1comprising the amino acid sequence selected from SEQ ID NO: 13 and SEQID NO: 25; (b) an HVR-H2 comprising the amino acid sequence of SEQ IDNO: 14; (c) an HVR-H3 comprising the amino acid sequence selected fromSEQ ID NO: 15 and SEQ ID NO: 20; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 16; (e) an HVR-L2 comprising the amino acidsequence selected from SEQ ID NO: 17, SEQ ID NO; 21 and SEQ ID NO: 23;and (f) an HVR-L3 comprising the amino acid sequence selected from SEQID NO: 18, SEQ ID NO: 22 and SEQ ID NO: 24.

In one aspect, the invention provides an antibody comprising a heavychain variable domain selected from SEQ ID NO: 6, SEQ ID NO: 8, SEQ IDNO: 10, SEQ ID NO: 12. In one aspect, the invention provides an antibodycomprising a light chain variable domain selected from SEQ ID NO: 5, SEQID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11. In one aspect, the inventionprovides an antibody comprising a heavy chain variable domain comprisingSEQ ID NO: 6. In one aspect, the invention provides an antibodycomprising a light chain variable domain comprising SEQ ID NO: 5. In oneaspect, the invention provides an antibody comprising a heavy chainvariable domain comprising SEQ ID NO: 6 and a light chain variabledomain comprising SEQ ID NO: 5. In one aspect, the invention provides anantibody comprising a heavy chain variable domain comprising SEQ ID NO:8. In one aspect, the invention provides an antibody comprising a lightchain variable domain comprising SEQ ID NO: 7. In one aspect, theinvention provides an antibody comprising a heavy chain variable domaincomprising SEQ ID NO: 8 and a light chain variable domain comprising SEQID NO: 7. In one aspect, the invention provides an antibody comprising aheavy chain variable domain comprising SEQ ID NO: 10. In one aspect, theinvention provides an antibody comprising a light chain variable domaincomprising SEQ ID NO: 9. In one aspect, the invention provides anantibody comprising a heavy chain variable domain comprising SEQ ID NO:10 and a light chain variable domain comprising SEQ ID NO: 9. In oneaspect, the invention provides an antibody comprising a heavy chainvariable domain comprising SEQ ID NO: 12. In one aspect, the inventionprovides an antibody comprising a light chain variable domain comprisingSEQ ID NO: 11. In one aspect, the invention provides an antibodycomprising a heavy chain variable domain comprising SEQ ID NO: 12 and alight chain variable domain comprising SEQ ID NO: 11.

In one aspect, the invention provides an anti-Factor D antibodycomprising a heavy chain variable domain comprising an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 6, 8, 10 and 12. In some embodiments, an aminoacid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% sequence identity contains substitutions, insertions, ordeletions relative to the reference sequence, but an antibody comprisingthat amino acid sequence retains the ability to bind to Factor D. Insome embodiments, a total of 1 to 10 amino acids have been substituted,inserted, or deleted in a sequence selected from the group consisting ofSEQ ID NO: 6, 8, 10 or 12. In some embodiments, the substitutions,insertions or deletions occur in regions outside the HVRs (i.e., in theFRs). In some embodiments, an anti-Factor D antibody comprises a heavychain variable domain comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 6, 8, 10 or 12.

In some embodiments, the invention provides an anti-Factor D antibodycomprising a light chain variable domain comprising an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 5, 7, 9 and 11. In some embodiments, an aminoacid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% sequence identity contains substitutions, insertions, ordeletions relative to the reference sequence, but an antibody comprisingthat amino acid sequence retains the ability to bind to Factor D. Insome embodiments, a total of 1 to 10 amino acids have been substituted,inserted, or deleted in a sequence selected from the group consisting ofSEQ ID NO: 5, 7, 9 and 11. In some embodiments, the substitutions,insertions or deletions occur in regions outside the HVRs (i.e., in theFRs). In some embodiments, an anti-Factor D antibody comprises a lightchain variable domain comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 5, 7, 9 and 11.

An anti-Factor D antibody may comprise any suitable framework variabledomain sequence, provided that the antibody retains the ability to bindFactor D. For example, in some embodiments, anti-Factor D antibodies ofthe invention comprise a heavy chain variable domain framework sequencethat is a combination of VI.4.1 b+ and JH4d (See FIG. 3). In someembodiments, anti-Factor D antibodies of the invention comprise a humansubgroup VII heavy chain framework consensus sequence. In someembodiments, anti-Factor D antibodies of the invention comprise a heavychain variable domain framework sequence comprising FR1 comprising aminoacids 1-25 of SEQ ID NO: 8, FR2 comprising amino acids 36-46 of SEQ IDNO: 8, FR3 comprising amino acids 67-98 of SEQ ID NO: 8 and FR4comprising amino acids 105-115 of SEQ ID NO: 8 In one embodiment ofthese antibodies, the heavy chain variable domain sequence comprisessubstitution(s) at position 40 and/or 88 (Kabat numbering). In oneembodiment of these antibodies, position 40 is cysteine (C) or alanine(A) and/or position 88 is cysteine (C) or alanine (A). In someembodiments, anti-Factor D antibodies of the invention comprise a lightchain variable domain framework sequence that is a combination of DPK4and JK2 (See FIG. 4). In some embodiments, anti-Factor D antibodies ofthe invention comprise a human kappa I (κI) light chain frameworkconsensus sequence. In some embodiments, anti-Factor D antibodies of theinvention comprise a light chain variable domain framework sequencecomprising FR1 comprising amino acids 1-23 of SEQ ID NO: 7, FR2comprising amino acids 35-49 of SEQ ID NO: 7, FR3 comprising amino acids57-88 of SEQ ID NO: 7 and FR4 comprising amino acids 98-107 of SEQ IDNO: 7. In one embodiment of these antibodies, the light chain variableframework sequence comprises one or more substitution(s) at position 15,43 and/or 104 (Kabat numbering). In one embodiment of these antibodies,position 15 is cysteine (C) or valine (V), position 43 is cysteine (C)or alanine (A) and/or position 104 is valine (V) or leucine (L).

Further, an anti-Factor D antibody may comprise any suitable constantdomain sequence, provided that the antibody retains the ability to bindFactor D. For example, in some embodiments, anti-Factor D antibodies ofthe invention comprise at least a portion of a heavy chain constantdomain. In one embodiment, anti-Factor D antibodies of the inventioncomprise a heavy chain constant domain of either one or a combination ofan α, δ, ε, γ, or μ heavy chain. Depending on the amino acid sequence ofthe constant domain of their heavy chains (C_(H)), immunoglobulins canbe assigned to different classes or isotypes. There are five classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chainsdesignated α, δ, ε, γ, and μ, respectively. The γ and α classes arefurther divided into subclasses on the basis of relatively minordifferences in C_(H) sequence and function, e.g., humans express thefollowing subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In oneembodiment, anti-Factor D antibodies of the invention comprise a heavychain constant domain comprising substitutions at amino acid positionsthat results in a desired effect on effector function (e.g., bindingaffinity). In one embodiment, anti-Factor D antibodies of the inventioncomprise a heavy chain constant domain comprising substitutions at aminoacid positions that do not result in an effect on effector function(e.g., binding affinity). In one embodiment, anti-Factor D antibodies ofthe invention comprise a heavy chain constant domain of the IgG type(e.g. IgG1, IgG2, IgG3 or IgG4) and further comprise a substitution atposition 114 (Kabat numbering; equivalent to 118 in EU numbering), 168(Kabat numbering; equivalent to 172 in EU numbering), 172 (Kabatnumbering; equivalent to 176 in EU numbering) and/or 228 (EU numbering).In one embodiment, anti-Factor D antibodies of the invention comprise aheavy chain constant domain of the IgG (e.g. IgG1, IgG2, IgG3 or IgG4)type and further comprise a substitution at position 114 whereinposition 114 is a cysteine (C) or alanine (A), position 168 is cysteine(C) or alanine (A), position 172 is a cysteine (C) or alanine (A) and/orposition 228 is a proline (P), arginine (R) or serine (S).

Further, for example, in some embodiments, anti-Factor D antibodies ofthe invention comprise at least a portion of a light chain constantdomain. In one embodiment, anti-Factor D antibodies of the inventioncomprise a light chain constant domain of either one or a combination ofa kappa or a lambda light chain, as the light chain from any vertebratespecies can be assigned to one of two dearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains. In one embodiment, anti-Factor D antibodies of the inventioncomprise a light chain constant domain comprising substitutions at aminoacid positions that results in a desired effect on effector function(e.g., binding affinity). In one embodiment, anti-Factor D antibodies ofthe invention comprise a light chain constant domain comprisingsubstitutions at amino acid positions that do not result in an effect oneffector function (e.g., binding affinity). In one embodiment,anti-Factor D antibodies of the invention comprise a light chainconstant domain of the kappa type and further comprise a substitution atposition 110, 144, 146 and/or 168 (Kabat numbering). In one embodiment,anti-Factor D antibodies of the invention comprise a light chainconstant domain of the kappa type and further comprise a substitution atposition 110 wherein 110 is a cysteine (C) or valine (V), at position144 wherein 144 is a cysteine (C) or alanine (A), at position 146wherein 146 is a isoleucine (I) or valine (V) and/or at position 168wherein 168 is a cysteine (C) or serine (S).

In one aspect, the invention provides antibodies that compete withmurine antibody 166-32 and/or humanized anti-Factor D antibody clone#56, #111, #250 or #416, and/or an antibody comprising variable domainor HVR sequences of humanized anti-Factor D antibody clone #56, #111,#250 or #416. Antibodies that bind to the same epitope as murineantibody 166-32 and/or humanized anti-Factor D antibody clone #56, #111,#250 or #416, and/or an antibody comprising variable domain or HVRsequences of humanized anti-Factor D antibody clone #56, #111, #250 or#416 are also provided.

In one embodiment, the invention provides an anti-Factor D antibodywherein the monovalent affinity of the antibody to Factor D (e.g.,affinity of the antibody as a Fab fragment to Factor D) is lower, forexample at least 1-fold or 2-fold lower than the monovalent affinity ofa chimeric antibody (e.g. affinity of the cihmeric antibody as a Fabfragment to Factor D), comprising, consisting or consisting essentiallyof a light chain variable domain of SEQ ID NO: 2 and heavy chainvariable domain of SEQ ID NO: 1.

In one embodiment, the invention provides an anti-Factor D antibodywherein the bivalent affinity of the antibody to Factor D (e.g.,affinity of the antibody as an IgG to Factor D) is lower, for example atleast 1-fold or 2-fold lower than the bivalent affinity of a chimericantibody (e.g. affinity of the cihmeric antibody as an IgG to Factor D),comprising, consisting or consisting essentially of a light chainvariable domain of SEQ ID NO: 2 and heavy chain variable domain of SEQID NO: 1.

In another embodiment, the invention provides an anti-Factor D antibodywherein the monovalent affinity of the antibody to FactorD (e.g.,affinity of the antibody as a Fab fragment to Factor D) is greater, forexample at least 1-fold or 2-fold greater than the monovalent affinityof a chimeric antibody (e.g. affinity of the chimeric antibody as a Fabfragment to Factor D), comprising, consisting or consisting essentiallyof a light chain variable domain of SEQ ID NO: 2 and heavy chainvariable domain of SEQ ID NO: 1.

In another embodiment, the invention provides an anti-Factor D antibodywherein the bivalent affinity of the antibody to FactorD (e.g., affinityof the antibody as an IgG to Factor D) is greater, for example at least1-fold or 2-fold greater than the bivalent affinity of a chimericantibody (e.g. affinity of the chimeric antibody as an IgG to Factor D),comprising, consisting or consisting essentially of a light chainvariable domain of SEQ ID NO: 2 and heavy chain variable domain of SEQID NO: 1.

In another embodiment, the invention provides an anti-Factor D antibodywherein the affinity of the antibody in its monovalent form to Factor D(e.g., affinity of the antibody as a Fab fragment to Factor D) is 1.0 nM(1.0×10⁻⁹ M) or better. In another embodiment, the invention provides ananti-Factor D antibody wherein the affinity of the antibody in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) is 0.5 nM (0.5×10⁻⁹ M) or better. In anotherembodiment, the invention provides an anti-Factor D antibody wherein theaffinity of the antibody in its monovalent form to Factor D (e.g.,affinity of the antibody as a Fab fragment to Factor D) is 1.0 pM(1.0×10⁻¹² M) or better. In another embodiment, the invention providesan anti-Factor D antibody wherein the affinity of the antibody in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) is 0.5 pM (0.5×10⁻¹² M) or better.

In another embodiment, the invention provides an anti-Factor D antibodywherein the affinity of the antibody in its bivalent form to Factor D(e.g., affinity of the antibody as an IgG to Factor D) is 1.0 nM(1.0×10⁻⁹ M) or better. In another embodiment, the invention provides ananti-Factor D antibody wherein the affinity of the antibody in itsbivalent form to Factor D (e.g., affinity of the antibody as an IgG toFactor D) is 0.5 nM (0.5×10⁻⁹ M) or better. In another embodiment, theinvention provides an anti-Factor D antibody wherein the affinity of theantibody in its bivalent form to Factor D (e.g., affinity of theantibody as an IgG to Factor D) is 1.0 pM (1.0×10⁻¹² M) or better. Inanother embodiment, the invention provides an anti-Factor D antibodywherein the affinity of the antibody in its bivalent form to Factor D(e.g., affinity of the antibody as an IgG to Factor D) is 0.5 pM(0.5×10⁻¹² M) or better.

In another embodiment, the invention provides an anti-Factor D antibodywherein the affinity of the antibody in its monovalent form to Factor D(e.g., affinity of the antibody as a Fab fragment to Factor D) isbetween 0.5 mM (0.5×10⁻⁶ M) and 0.5 pM (0.5×10⁻¹² M). In anotherembodiment, the invention provides an anti-Factor D antibody wherein theaffinity of the antibody in its monovalent form to Factor D (e.g.,affinity of the antibody as a Fab fragment to Factor D) is between 15 nM(15×10⁻⁹M) and 0.1 nM (0.1×10⁻⁹ M). In another embodiment, the inventionprovides an anti-Factor D antibody wherein the affinity of the antibodyin its monovalent form to Factor D (e.g., affinity of the antibody as aFab fragment to Factor D) is between 5.5 nM (5.5×10⁻⁹ M) and 1 nM(1×10⁻⁹ M). In another embodiment, the invention provides an anti-FactorD antibody wherein the affinity of the antibody in its monovalent formto Factor D (e.g., affinity of the antibody as a Fab fragment to FactorD) is between 0.5 pM (0.5×10⁻¹² M) and 2 pM (2×10⁻¹² M).

In another embodiment, the invention provides an anti-Factor D antibodywherein the affinity of the antibody in its bivalent form to Factor D(e.g., affinity of the antibody as an IgG to Factor D) is between 0.5 mM(0.5×10⁻⁶ M) and 0.5 pM (0.5×10⁻¹² M). In another embodiment, theinvention provides an anti-Factor D antibody wherein the affinity of theantibody in its bivalent form to Factor D (e.g., affinity of theantibody as an IgG to Factor D) is between 10 nM (10×10⁻⁹ M) and 0.05 nM(0.05×10⁻⁹ M). In another embodiment, the invention provides ananti-Factor D antibody wherein the affinity of the antibody in itsbivalent form to Factor D (e.g., affinity of the antibody as an IgG toFactor D) is between 5.5 nM (5.5×10⁻⁹ M) and 1 nM (1×10⁻⁹). In anotherembodiment, the invention provides an anti-Factor D antibody wherein theaffinity of the antibody in its bivalent form to Factor D (e.g.,affinity of the antibody as an IgG to Factor D) is between 0.5 pM(0.5×10⁻¹² M) and 2 pM (2×10⁻¹² M).

In another embodiment, the invention provides an anti-Factor D antibodywherein the affinity of the antibody in its monovalent form to Factor D(e.g., affinity of the antibody as a Fab fragment to Factor D) is about3.7 nM (3.7×10⁻⁹ M). In another embodiment, the invention provides ananti-Factor D antibody wherein the affinity of the antibody in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) is about 3.3 nM (3.3×10⁻⁹ M). In anotherembodiment, the invention provides an anti-Factor D antibody wherein theaffinity of the antibody in its monovalent form to Factor D (e.g.,affinity of the antibody as a Fab fragment to Factor D) is about 5.1 nM(5.1×10⁻⁹ M). In another embodiment, the invention provides ananti-Factor D antibody wherein the affinity of the antibody in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) is about 2.7 nM (2.7×10⁻⁹ M). In anotherembodiment, the invention provides an anti-Factor D antibody wherein theaffinity of the antibody in its monovalent form to Factor D (e.g.,affinity of the antibody as a Fab fragment to Factor D) is about 1.4 nM(1.4×10⁻⁹ M). In another embodiment, the invention provides ananti-Factor D antibody wherein the affinity of the antibody in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) is about 1.4 pM (1.4×10⁻¹² M). In anotherembodiment, the invention provides an anti-Factor D antibody wherein theaffinity of the antibody in its bivalent form to Factor D (e.g.,affinity of the antibody as a IgG to Factor D) is about 1.1 pM (1×10⁻¹²M). In another embodiment, the invention provides an anti-Factor Dantibody wherein the affinity of the antibody in its monovalent form toFactor D (e.g., affinity of the antibody as a Fab fragment to Factor D)is about 0.19 nM (0.19×10⁻⁹). In another embodiment, the inventionprovides an anti-Factor D antibody wherein the affinity of the antibodyin its bivalent form to Factor D (e.g., affinity of the antibody as aIgG to Factor D) is about 0.08 nM (0.08×10⁻⁹ M). In another embodiment,the invention provides an anti-Factor D antibody wherein the affinity ofthe antibody in its monovalent form to Factor D (e.g., affinity of theantibody as a Fab fragment to Factor D) is about 12.3 nM (12.3×10⁻⁹ M).In another embodiment, the invention provides an anti-Factor D antibodywherein the affinity of the antibody in its bivalent form to Factor D(e.g., affinity of the antibody as a IgG to Factor D) is about 9.0 nM(9.0×10⁻⁹ M).

In another embodiment, the invention provides an anti-Factor D antibodywherein the affinity of the antibody in its monovalent form to Factor D(e.g., affinity of the antibody as a Fab fragment to Factor D) is about1.4 pM (1.4×10⁻¹² M)+/−0.5. In another embodiment, the inventionprovides an anti-Factor D antibody wherein the affinity of the antibodyin its bivalent form to Factor D (e.g., affinity of the antibody as anIgG to Factor D) is about 1.1 pM (1.1×10⁻¹²M)+/−0.6. In anotherembodiment, the invention provides an anti-Factor D antibody wherein theaffinity of the antibody in its monovalent form to Factor D (e.g.,affinity of the antibody as a Fab fragment to Factor D) is about 0.19 nM(0.19×10⁻⁹ M)+/−0.01. In another embodiment, the invention provides ananti-Factor D antibody wherein the affinity of the antibody in itsbivalent form to Factor D (e.g., affinity of the antibody as a IgG toFactor D) is about 0.08 nM (0.08×10⁻⁹ M)+/−0.01. In another embodiment,the invention provides an anti-Factor D antibody wherein the affinity ofthe antibody in its monovalent form to Factor D (e.g., affinity of theantibody as a Fab fragment to Factor D) is about 12.3 nM (12.3×10⁻⁹M)+/−2. In another embodiment, the invention provides an anti-Factor Dantibody wherein the affinity of the antibody in its bivalent form toFactor D (e.g., affinity of the antibody as a IgG to Factor D) is about9.0 nM (9.0×10⁻⁹ M)+/−1.

In another embodiment, an anti-Factor D antibody may have an affinity inits monovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) of about 3.7 nM (3.7×10⁻⁹ M)+/−2. In anotherembodiment, an anti-Factor D antibody may have an affinity in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) of about 3.3 nM (3.3×10⁻⁹ M)+/−2. In anotherembodiment, an anti-Factor D antibody may have an affinity in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) of about 5.1 nM (5.1×10⁻⁹M)+/−2. In anotherembodiment, an anti-Factor D antibody may have an affinity in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) of about 2.7 nM (2.7×10⁻⁹ M)+/−2. In anotherembodiment, an anti-Factor D antibody may have an affinity in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) of about 1.4 nM (1.4×10⁻⁹ M)+/−2. In anotherembodiment, an anti-Factor D antibody may have an affinity in itsmonovalent form to Factor D (e.g., affinity of the antibody as a Fabfragment to Factor D) of about 1.4 pM (1.4×10⁻¹² M)+/−2. In anotherembodiment, an anti-Factor D antibody may have an affinity in itsbivalent form to Factor D (e.g., affinity of the antibody as a IgG toFactor D) of about 1.1 pM (1.1×10⁻¹² M)+/−2. In another embodiment, ananti-Factor D antibody may have an affinity in its monovalent form toFactor D (e.g., affinity of the antibody as a Fab fragment to Factor D)is about 0.19 nM (0.19×10⁻⁹ M)+/−2. In another embodiment, ananti-Factor D antibody may have an affinity in its bivalent form toFactor D (e.g., affinity of the antibody as a IgG to Factor D) is about0.08 nM (0.08×10⁻⁹ M)+/−2. In another embodiment, an anti-Factor Dantibody may have an affinity in its monovalent form to Factor D (e.g.,affinity of the antibody as a Fab fragment to Factor D) is about 12.3 nM(12.3×10⁻⁹ M)+/−2. In another embodiment, an anti-Factor D antibody mayhave an affinity in its bivalent form to Factor D (e.g., affinity of theantibody as a IgG to Factor D) is about 9.0 nM (9.0×10⁻⁹ M)+/−2.

As is well-established in the art, binding affinity of a ligand to itsreceptor can be determined using any of a variety of assays, andexpressed in terms of a variety of quantitative values. Accordingly, inone embodiment, the binding affinity is expressed as Kd values andreflects intrinsic binding affinity (e.g., with minimized avidityeffects). Generally and preferably, binding affinity is measured invitro, whether in a cell-free or cell-associated setting. As describedin greater detail herein, fold difference in binding affinity can bequantified in terms of the ratio of the monovalent binding affinityvalue of a humanized antibody (e.g., in Fab form) and the monovalentbinding affinity value of a reference/comparator antibody (e.g., in Fabform) (e.g., a murine antibody having donor hypervariable regionsequences), wherein the binding affinity values are determined undersimilar assay conditions. Thus, in one embodiment, the fold differencein binding affinity is determined as the ratio of the Kd values of thehumanized antibody in Fab form and said reference/comparator Fabantibody. For example, in one embodiment, if an antibody of theinvention (A) has an affinity that is “3-fold lower” than the affinityof a reference antibody (M), then if the Kd value for A is 3×, the Kdvalue of M would be 1×, and the ratio of Kd of A to Kd of M would be3:1. Conversely, in one embodiment, if an antibody of the invention (C)has an affinity that is “3-fold greater” than the affinity of areference antibody (R), then if the Kd value for C is 1×, the Kd valueof R would be 3×, and the ratio of Kd of C to Kd of R would be 1:3. Anyof a number of assays known in the art, including those describedherein, can be used to obtain binding affinity measurements, including,for example, Biacore, radioimmunoassay (RIA) and ELISA.

Further, Kd values for an antibody of the invention may vary dependingon conditions of the particular assay used. For example, in oneembodiment, binding affinity measurements may be obtained in an assaywherein the Fab or antibody is immobilized and binding of the ligand,i.e. Factor D, is measured or alternatively, the ligand, i.e. Factor D,for the Fab or antibody is immobilized and binding of the Fab orantibody is measured. In one embodiment, the binding affinitymeasurements may be obtained in an assay wherein the regenerationconditions may comprise (1) 10 mM glycein or 4M MgCl₂ at pH 1.5, and (2)pH between pH of 1.0 and pH of 7.5, including pH of 1.5, pH of 5.0, pHof 6.0 and pH of 7.2. In one embodiment, the binding affinitymeasurements may be obtained in an assay wherein the binding conditionsmay comprise (1) PBS or HEPES-buffered saline and (2) Tween-20, i.e.0.1% Tween-20. In one embodiment, the binding affinity measurements maybe obtained in an assay wherein the source of the ligand, i.e. Factor D,may be from commercially available sources. In one embodiment, bindingaffinity measurements may be obtained in an assay wherein (1) the Fab orantibody is immobilized and binding of the ligand, i.e. Factor D ismeasured, (2) the regeneration conditions comprise 4M MgCl₂ at pH 7.2and (3) the binding conditions comprise HEPES-buffered saline, pH 7.2containing 0.1% Tween-20. In one embodiment, binding affinitymeasurements may be obtained in an assay wherein (1) the ligand, i.e.Factor D, is immobilized and binding of the Fab or antibody is measured,(2) the regeneration conditions comprise 10 mM glycine at pH 1.5 and (3)the binding conditions comprise PBS buffer.

The terms “cell”, “cell line” and “cell culture” include progeny. It isalso understood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Variant progenythat have the same function or biological property, as screened for inthe originally transformed cell, are included. The “host cells” used inthe present invention generally are prokaryotic or eukaryotic hosts.

The term “vector” means a DNA construct containing a DNA sequence whichis operably linked to a suitable control sequence capable of effectingthe expression of the DNA in a suitable host. Such control sequencesinclude a promoter to effect transcription, an optional operatorsequence to control such transcription, a sequence encoding suitablemRNA ribosome binding sites, and sequences which control the terminationof transcription and translation. The vector may be a plasmid, a phageparticle, or simply a potential genomic insert. Once transformed into asuitable host, the vector may replicate and function independently ofthe host genome, or may in some instances, integrate into the genomeitself. In the present specification, “plasmid” and “vector” aresometimes used interchangeably, as the plasmid is the most commonly usedform of vector. However, the invention is intended to include such otherforms of vectors which serve equivalent function as and which are, orbecome, known in the art.

The word “label” when used herein refers to a detectable compound orcomposition which can be conjugated directly or indirectly to a moleculeor protein, e.g., an antibody. The label may itself be detectable (e.g.,radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

As used herein, “solid phase” means a non-aqueous matrix to which theantibody of the present invention can adhere. Example of solid phasesencompassed herein include those formed partially or entirely of glass(e.g. controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g. an affinity chromatography column).

Generation of Antibodies Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are prokaryotic, yeast, or higher eukaryotic cells. Suitableprokaryotes for this purpose include both Gram-negative andGram-positive organisms, for example, Enterobacteria such as E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia, andShigella, as well as Bacilli, Pseudomonas, and Streptomyces. Onepreferred E. coli cloning host is E. coli 294 (ATCC 31,446), althoughother strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E.coli W3110 (ATCC 27,325) are suitable. These examples are illustrativerather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae is the most commonlyused among lower eukaryotic host microorganisms. However, a number ofother genera, species, and strains are commonly available and usefulherein, such as Schizosaccharomyces pombe; Kluyveromyces; Candida;Trichoderma; Neurospora crassa; and filamentous fungi such as e.g.,Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such asA. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibodies arederived from multicellular organisms. In principal, any highereukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells, Luckow et al., Bio/Technology 6, 47-55 (1988); Miller etal., Genetic Engineering, Setlow et al. eds. Vol. 8, pp. 277-279 (Plenampublishing 1986); Mseda et al., Nature 315, 592-594 (1985). Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori havebeen identified. A variety of viral strains for transfection arepublicly available, e.g., the L-1 variant of Autographa californica NPVand the Bm-5 strain of Bombyx mori NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Moreover, plant cellscultures of cotton, corn, potato, soybean, petunia, tomato, and tobaccoand also be utilized as hosts.

Vertebrate cells, and propagation of vertebrate cells, in culture(tissue culture) has become a routine procedure. See Tissue Culture,Academic Press, Kruse and Patterson, eds. (1973). Examples of usefulmammalian host cell lines are monkey kidney; human embryonic kidneyline; baby hamster kidney cells; Chinese hamster ovary cells/-DHFR (CHO,Urtaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); mousesertoli cells; human cervical carcinoma cells (HELA); canine kidneycells; human lung cells; human liver cells; mouse mammary tumor; and NS0cells.

Host cells are transformed with the above-described vectors for antibodyproduction and cultured in conventional nutrient media modified asappropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences.

The host cells used to produce the antibody variant of this inventionmay be cultured in a variety of media. Commercially available media suchas Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) aresuitable for culturing host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enzymol. 58: 44 (1979), Barnes et al.,Anal. Biochem. 102: 255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866;4,560,655; 5,122,469; 5,712,163; or 6,048,728 may be used as culturemedia for the host cells. Any of these media may be supplemented asnecessary with hormones and/or other growth factors (such as Insulin,transferrin, or epidermal growth factor), salts (such as X-chlorides,where X is sodium, calcium, magnesium; and phosphates), buffers (such asHEPES), nucleotides (such as adenosine and thymidine), antibiotics (suchas GENTAMYCIN™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

Antibody Purification

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody variant is produced intracellularly, as a firststep, the particulate debris, either host cells or lysed fragments, maybe removed, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10: 163-167 (1992) describe a procedure forisolating antibodies which are secreted to the periplasmic space of E.coli. Briefly, cell paste is thawed in the presence of sodium acetate(pH 3.5), EDTA, and phenylmethylsulfonyifluoride (PMSF) over about 30minutes. Cell debris can be removed by centrifugation. Where theantibody variant is secreted into the medium, supernatants from suchexpression systems are generally first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. A protease inhibitor such asPMSF may be included in any of the foregoing steps to inhibitproteolysis and antibiotics may be included to prevent the growth ofadventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel elecrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody variant.Protein A can be used to purify antibodies that are based on human IgG1,IgG2 or IgG4 heavy chains (Lindmark et al., J. Immunol Meth. 62: 1-13(1983)). Protein G is recommended for all mouse isotypes and for humanIgG3 (Guss et al., EMBO J. 5: 1567-1575 (1986)). The matrix to which theaffinity ligand is attached is most often agarose, but other matricesare available. Mechanically stable matrices such as controlled poreglass or poly(styrenedivinyl)benzene allow for faster flow rates andshorter processing times than can be achieved with agarose. Where theantibody variant comprises a CH3 domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, Reverse Phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE™ chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody variant to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody variant of interest and contaminants may be subjected tolow pH hydrophobic interaction chromatography using an elution buffer ata pH between about 2.5-4.5, preferably performed at low saltconcentrations (e.g., from about 0-0.25M salt).

Pharmaceutical Formulations

Therapeutic formulations of the polypeptide or antibody may be preparedfor storage as lyophilized formulations or aqueous solutions by mixingthe polypeptide having the desired degree of purity with optional“pharmaceutically-acceptable” carriers, excipients or stabilizerstypically employed in the art (all of which are termed “excipients”).For example, buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic detergents, antioxidants and other miscellaneousadditives. (See Remington's Pharmaceutical Sciences, 16th edition, A.Osol, Ed. (1980)). Such additives must be nontoxic to the recipients atthe dosages and concentrations employed.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are preferably present at concentrationranging from about 2 mM to about 50 mM. Suitable buffering agents foruse with the present invention include both organic and inorganic acidsand salts thereof such as citrate buffers (e.g., monosodiumcitrate-disodium citrate mixture, citric acid-trisodium citrate mixture,citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additionally, there may be mentioned phosphatebuffers, histidine buffers and trimethylamine salts such as Tris.

Preservatives may be added to retard microbial growth, and may be addedin amounts ranging from 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalconium halides (e.g., chloride, bromide, iodide),hexamethonium chloride, alkyl parabens such as methyl or propyl paraben,catechol, resorcinol, cyclohexanol, and 3-pentanol.

Isotonicifiers sometimes known as “stabilizers” may be added to ensureisotonicity of liquid compositions of the present invention and includepolhydric sugar alcohols, preferably trihydric or higher sugar alcohols,such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, omithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol,.alpha.-monothioglycerol and sodium thio sulfate; low molecular weightpolypeptides (i.e. <10 residues); proteins such as human serum albumin,bovine serum albumin, gelatin or immunoglobulins; hydrophylic polymers,such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose,fructose, glucose; disaccharides such as lactose, maltose, sucrose andtrisaccacharides such as raffinose; polysaccharides such as dextran.Stabilizers may be present in the range from 0.1 to 10,000 weights perpart of weight active protein.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe added to help solubilize the therapeutic agent as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stressedwithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers(Tween®-20, Tween®-80, etc.). Non-ionic surfactants may be present in arange of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07mg/ml to about 0.2 mg/ml.

Additional miscellaneous excipients include bulking agents, (e.g.starch), chelating agents (e.g. EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), and cosolvents. The formulation herein mayalso contain more than one active compound as necessary for theparticular indication being treated, preferably those with complementaryactivities that do not adversely affect each other. For example, it maybe desirable to further provide an immunosuppressive agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended. The active ingredients may also beentrapped in microcapsule prepared, for example, by coacervationtechniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin micropheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, A. Osal, Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished, for example, by filtration through sterilefiltration membranes. Sustained-release preparations may be prepared.Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing theantibody variant, which matrices are in the form of shaped articles,e.g., films, or microcapsules. Examples of sustained-release matricesinclude polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C. resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS-S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The amount of therapeutic polypeptide, antibody or fragment thereofwhich will be effective in the treatment of a particular disorder orcondition will depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques. Where possible, it isdesirable to determine the dose-response curve and the pharmaceuticalcompositions of the invention first in vitro, and then in useful animalmodel systems prior to testing in humans.

In a preferred embodiment, an aqueous solution of therapeuticpolypeptide, antibody or fragment thereof is administered bysubcutaneous injection. Each dose may range from about 0.5 μg to about50 μg per kilogram of body weight, or more preferably, from about 3 μgto about 30 μg per kilogram body weight.

The dosing schedule for subcutaneous administration may vary form once amonth to daily depending on a number of clinical factors, including thetype of disease, severity of disease, and the subject's sensitivity tothe therapeutic agent.

Uses for the Humanized Antibody

The humanized antibodies of the present invention are useful indiagnostic assays, e.g., for detecting expression of a target ofinterest in specific cells, tissues, or serum. For diagnosticapplications, the antibody variant typically will be labeled with adetectable moiety. Numerous labels are available. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,.beta.-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for Use inEnzyme Immunoassay, in Methods in Enzym. (Ed. J. Langone & H. VanVunakis), Academic press, New York, 73: 147-166 (1981).

Sometimes, the label is indirectly conjugated with the antibody variant.The skilled artisan will be aware of various techniques for achievingthis. For example, the antibody variant can be conjugated with biotinand any of the three broad categories of labels mentioned above can beconjugated with avidin, or vice versa. Biotin binds selectively toavidin and thus, the label can be conjugated with the antibody variantin this indirect manner. Alternatively, to achieve indirect conjugationof the label with the antibody variant, the antibody variant isconjugated with a small hapten (e.g. digloxin) and one of the differenttypes of labels mentioned above is conjugated with an anti-haptenantibody variant (e.g. anti-digloxin antibody). Thus, indirectconjugation of the label with the antibody variant can be achieved.

In another embodiment of the invention, the antibody variant need not belabeled, and the presence thereof can be detected using a labeledantibody which binds to the antibody variant.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample for binding with a limited amount ofantibody variant. The amount of target in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition. As a result, the standard and test sample thatare bound to the antibodies may conveniently be separated from thestandard and test sample which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, or the proteinto be detected. In a sandwich assay, the test sample to be analyzed isbound by a first antibody which is immobilized on a solid support, andthereafter a second antibody binds to the test sample, thus forming aninsoluble three-part complex. See e.g., U.S. Pat. No. 4,376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody that is labeled with a detectable moiety (indirect sandwichassay). For example, one type of sandwich assay is an ELISA assay, inwhich case the detectable moiety is an enzyme.

For immunohistochemistry, the tumor sample may be fresh or frozen or maybe embedded in paraffin and fixed with a preservative such as formalin,for example.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody variant is labeled with a radionucleotide (suchas .sup.111 In, .sup.99 Tc, .sup.14 C, .sup.131 I, .sup.3 H, .sup.32 Por .sup.35 S) so that the tumor can be localized usingimmunoscintiography. For example, a high affinity anti-IgE antibody ofthe present invention may be used to detect the amount of IgE presentin, e.g., the lungs of an asthmatic patient.

The antibody of the present invention can be provided in a kit, i.e.,packaged combination of reagents in predetermined amounts withinstructions for performing the diagnostic assay. Where the antibodyvariant is labeled with an enzyme, the kit may include substrates andcofactors required by the enzyme (e.g., a substrate precursor whichprovides the detectable chromophore or fluorophore). In addition, otheradditives may be included such as stabilizers, buffers (e.g., a blockbuffer or lysis buffer) and the like. The relative amounts of thevarious reagents may be varied widely to provide for concentrations insolution of the reagents which substantially optimize the sensitivity ofthe assay. Particularly, the reagents may be provided as dry powders,usually lyophilized, including excipients which on dissolution willprovide a reagent solution having the appropriate concentration.

In Vivo Uses for the Antibody

It is contemplated that the antibodies of the present invention may beused to treat a mammal. In one embodiment, the antibody is administeredto a nonhuman mammal for the purposes of obtaining preclinical data, forexample. Exemplary nonhuman mammals to be treated include nonhumanprimates, dogs, cats, rodents and other mammals in which preclinicalstudies are performed. Such mammals may be established animal models fora disease to be treated with the antibody or may be used to studytoxicity of the antibody of interest. In each of these embodiments, doseescalation studies may be performed on the mammal.

The antibody or polypeptide is administered by any suitable means,including parenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local immunosuppressive treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibody variant issuitably administered by pulse infusion, particularly with decliningdoses of the antibody variant. Preferably the dosing is given byinjections, most preferably intravenous or subcutaneous injections,depending in part on whether the administration is brief or chronic.

For the prevention or treatment of disease, the appropriate dosage ofthe antibody or polypeptide will depend on the type of disease to betreated, the severity and course of the disease, whether the antibodyvariant is administered for preventive or therapeutic purposes, previoustherapy, the patient's clinical history and response to the antibody,and the discretion of the attending physician.

Depending on the type and severity of the disease, about 0.1 mg/kg to150 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. A typicaldaily dosage might range from about 1 mg/kg to 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays. Anexemplary dosing regimen is disclosed in WO 94/04188.

The antibody compositions may be formulated, dosed and administered in amanner consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the antibody to be administeredwill be governed by such considerations, and is the minimum amountnecessary to prevent, ameliorate, or treat a disease or disorder. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as used hereinbefore or about from 1 to99% of the heretofore employed dosages.

The antibodies of the present invention which recognize Factor D astheir target may be used to treat complement-mediated disorders. Thesedisorders are associated with excessive or uncontrolled complementactivation. They include: Complement activation during cardiopulmonarybypass operations; complement activation due to ischemia-reperfusionfollowing acute myocardial infarction, aneurysm, stroke, hemorrhagicshock, crush injury, multiple organ failure, hypobolemic shock andintestinal ischemia. These disorders can also include disease orcondition is an inflammatory condition such as severe burns,endotoxemia, septic shock, adult respiratory distress syndrome,hemodialysis; anaphylactic shock, severe asthma, angioedema, Crohn'sdisease, sickle cell anemia, poststreptococcal glomerulonephritis andpancreatitis. The disorder may be the result of an adverse drugreaction, drug allergy, IL-2 induced vascular leakage syndrome orradiographic contrast media allergy. It also includes autoimmune diseasesuch as systemic lupus erythematosus, myasthenia gravis, rheumatoidarthritis, Alzheimer's disease and multiple sclerosis. Complementactivation is also associated with transplant rejection. Recently therehas been a strong correlation shown between complement activation andocular diseases such as age-related macular degeneration, diabeticretinopathy.

EXAMPLES

The following examples are offered by way of illustration and not by wayof limitation.

Example 1: Humanization of Factor D Murine MAb 166-32

The sequences of the heavy chain variable region (V_(H)) and the lightchain variable region (V_(L)) of murine mAb 166-32 were compared withhuman antibody germline sequences available in the public databases.Several criteria were used when deciding on a template as described instep 1 above, including overall length, similar CDR position within theframework, overall homology, size of the CDR, etc. All of these criteriataken together provided a result for choosing the optimal human templateas shown in the sequence alignment between 166-32 MAb heavy and lightchain sequences and the respective human template sequences depicted inFIGS. 3 and 4.

In this case, more than one human framework template was used to designthis antibody. The human template chosen for the V_(H) chain was acombination of VI-4.1b+ (7-04.1 locus)(access# X62110)(VH7 family) andJH4d (See FIG. 3). The human template chosen for the V_(L) chain was acombination of DPK4 (VK I family) combined with JK2 (See FIG. 4).

Once the template was chosen, a Fab library was constructed by DNAsynthesis and overlapping PCR. The library was composed of synthesizedMAb 166-32 CDRs synthesized with the respective chosen human templates.The overlapping nucleotides encoding partial V_(H) and V_(L) sequenceswere synthesized in the range of about 63 to about 76 nucleotides with18 to 21 nucleotide overlaps. Vectors expressing a library of humanizedFabs against Factor D antigen were constructed, and transformed into E.coli DH10B then plated on XL-1B bacterial lawn.

Library quality was evaluated for the size (the number of independentclones) and the diversity (the distribution of the mutations). Theindividual clones with double insertion of both light and heavy chainwas about 14 out of 20 sequenced. Framework wobble mutations were evenlydistributed.

PCR amplification of V₁ and V_(H) gene was performed using abiotinylated forward primer containing the specific sequence to theframework region FR1 and an overhanging sequence annealed to the end ofleader sequence (GeneIII) and a reverse primer from the conservedconstant region (Cκ or CH1) under standard PCR conditions. The PCRproduct was purified by agarose gel electrophoresis, or by commercialPCR purification kit to remove unincorporated biotinylated primers andnon-specific PCR.

Example 2: Library Screening

Capture Filter Lift was used for primary screening. The actual screeningsized is more than 3 times larger than the theoretic library size. Thecandidates were further screened by single-point ELISA assay. The bestbinders were further confirmed by direct antigen titration using FactorD based on Fab concentration

Capture Lift Screening

Capture Filter Lift Assay was used for primary screening for the bindingof Fab to Factor D. High titer phage were plated and incubated at 37° C.till use (about 6-8 hr). Goat anti-human kappa was diluted to 10 ug/mlin 10 ml PBST; Nitrocellulose filters for lifting plaques were preparedaccording to standard plaque lifting procedures and then immersed in 10ml blocking buffer for 2 hrs on a shaker. The filters were rinsed 3×with PBST. The filters were applied to a plaque lawn and incubated at RTfor approximately 15-24 hours. The filters were then removed from theplates and rinsed with TBST 3×.

Factor D (50 ug/ml) was diluted in PBST to 0.1 ug/ml and 4 ml per filterwas added. The filters were Incubated in the solution for 2 hr on ashaker at RT followed by rinsing 3×, each time 5 min. Diluted166-222-HRP (1:10,000 with PBST) was added at a volume of 4 ml perfilter and the filters were incubated for 1 hr on a shaker. The filterswere rinsed 4×. The filters were dried and then immersed in TMBsubstrate followed by immersion in water to stop the reaction. Positiveclones were identified.

Example 3: Single-Point ELISA Screening

Single-Point ELISA assay was used for the secondary screening. ImmulonII plates were coated with goat anti-human Fab (1:12,000, 50 ul/well)over night at RT. The next day the plates were washed 4× with a platewasher. Blocking buffer was added at a volume of 100 ul per well andplates incubated for 1 hr at RT. The plates were then washed 4×.

Each Fab to be screened was added at a volume of 50 ul per well (eitherfrom 15 ml periplasmic preparation or supernatant) and incubated 1 hr atRT. Plates were washed 4× followed by the addition of 50 ul/wellbiotinylated factor D at 0.01 ug/ml. Plates were incubated for 1 hr atRT and then washed 4×. StreptAvidin-HRP was added (1:10,000 in PBST) andincubated for 1 hr at RT. Plates were washed 5× and then developed byadding TMB substrate at 50 ul/well. Stop buffer was added at a volume of50 ul when it is well-developed (10-45 min) and the plates were read at450 nm.

Example 4: Sequencing of Humanized Anti-Factor D Clones

Sixteen humanized clones with good binding affinity for human Factor Dwere sequenced (see Table 1). Among these, position 2 (100% human) and49 (100% mouse) in the light chain, and position 93 (100% mouse) in theheavy chain are highly conserved indicating that they are important inmaintaining antibody binding ability.

TABLE 1 Amino acid sequence analysis of humanized clones from thehumanization library VK 2 4 13 43 49 64 69 VH 2 9 38 93 97 Mouse T V M PS S A I P K T E Human I M A V Y G T V S R V A 7 I M M V S S T I P K V E30 I V A V S S A V P K T E 45 I V M V S G A I S R V E 46 I V M V S S T IS R V E 47 I V A V S S T I S R V E 48 I V M V S S T V P R V E 50 I V M VS G A V P R T E 51 I M M V S G T I S K T E 56 I V A V S G T V P K T E 57I M M V S S A V S R V E 58 I V M P S G A V P R V E 59 I V A P S S T V PK V E 60 I V M P S G T V P R V E 63 I V M V S S T V S R T E 74 I V M V SS T I S R V E

Clone #56 was evaluated by BIAcore analysis and hemolytic inhibitionassay. BIAcore analysis showed that clone #56 has a similar affinity tohuman Factor D as chimeric 166-32 Fab (see Table 4). Hemolyticinhibition assay showed that clone #56 is somewhat more potent thanchimeric 166-32 Fab (see FIG. 6). Clone #56 contains two murine residuesin the framework of light chain and four murine residues in the heavychain. (see Table 1). Based on these results, further optimization wascarried out.

TABLE 2 Amino acid sequence analysis of optimized antibodies from thehumanization/CDR3s optimization library VK Positions 2 4 13 43 49 64 69CDR-L3 92 93 97 166-32 T V M P S S A D N T Human I M A V Y G T Template104 I V M P S S T D S T 109 I V A V S G A M N T 111 I V A V S G T D S T112 I V A V S G T D S T 114 I V A V S G T D C T 121 I V A V S S A D N T125 I V A P S S T D N T 130 I V A V S S T D N S VH Positions 2 9 38 9397 CDR-H3 98 99 100 166-32 I P K T E V D N Human Template V S R V A 104V S R V E V D T 109 V S R V E V N N 111 V S R V E V N N 112 V S R V E VN N 114 V S K V E V N N 121 I S R V E V N T 125 V S R V E P D N 130 V SR V E V D H

Clone #111 and #114 were characterized by BIAcore analysis (see Table4). Clone #104, #111, #114 and #130 were also characterized by hemolyticinhibition assay (see FIG. 6). These clones have higher affinities thanchimeric 166-32, and are more potent than chimeric Fab in inhibiting thealternative pathway as shown by hemolytic inhibition assay (FIG. 7).Clone #111 contains the same two murine residues in the light chain(position 4 and 49) as clone #56. It also contains the conserved murineresidue in the heavy chain position 97 as found in clone #56. There isone beneficial mutation in both light and heavy chain CDR3 in clone#111. From two independent libraries screened (humanization library, andhumanization/CDR3s optimization library), we found that the best cloneshave similar consensus residues.

To further optimize the affinity of clone #111, an antibody library wasconstructed by introducing single mutations into the CDR-H1 and CDR-L2simultaneously. In brief, site-directed mutagenesis approach was used toconstruct such libraries by annealing oligonucleotides encoding singlemutations to the template of clone #111. A total of 24 clones with veryhigh affinity to human Factor D were sequenced. Among those 24 clones,several redundant beneficial mutations were identified. Clones #250,#315, #345 and #416 were selected for BIAcore analysis (see Table 4).BIAcore data showed that these clones have higher affinity to humanFactor D than initial done #111. Clone #250, #315, #348 and #416 werealso tested in the hemolytic inhibition assay (see FIG. 6) andinhibition of the alternative pathway (FIG. 7).

Example 5: AP Hemolysis Assay

Biological function of the humanized clones was determined usinghemolytic inhibition assay and BIAcore analysis (See Example 6 below).Hemolytic assay was performed according to the following procedure. 20ul of 1:20 diluted rabbit red blood cells (RRBC) (0.5 ml+9.5 mlGVB/Mg-EGTA buffer) in 20 ml Saline (0.9% NaCl) at approximately 1:2×10⁴dilution were counted by Coulter Counter. The cell concentration wasthen adjusted to about 2-5×10⁴ cells/mi. Each plate received about500×10⁶/plate RRBC or about 1 ml RRBC/plate (500×10⁶/2-5×10⁴).

Cells were diluted in 6 ml GVB/Mg-EGTA buffer/plate, mixed and washed 3times by spinning at 1360 rpm×4 min at 4° C. The RRBC pellet issuspended in 3 ml GVB/Mg-EGTA buffer/plate and kept on ice.

Human serum from −80° C. freezer was thawed just prior to use. The serumwas diluted to a concentration of 20% serum in GVB/Mg-EGTA buffer, 5ml/plate (final is 10%) and kept on ice.

TABLE 3 1 2 3 S SB GVB/Mg-EGTA: 50 ul

50 ul

50 ul 50 ul 50 ul mAb: 50 ul mix 50 ul 50 ul 50 ul — — 20% Hu serum: 50ul 50 ul 50 ul 50 ul 50 ul Shake 30 sec at 5-6°, then keep at RT for 7min. Rabbit RBC: 30 ul 30 ul 30 ul 30 ul

Samples were shaken for 30 sec at 5-6° C. and then for 40 minutes at 37°C. Samples were cooled to 5-6° C. while shaking and then centrifuged at2,000 rpm×3 min at 4° C. Approximately 80 ul supernatant was transferredto a flat-bottom 96 well plate and the OD value at 590 nm was read usinga standard plate reader. The percent inhibition was calculated asfollows: % Inhibition={[(S−SB)−(U−SB)]/(S−SB)}×100%. (U=sample 1, 2 or 3(columns 1, 2 or 3 of Table 3, respectively).

Example 6: Kinetic Analysis of Anti-Human Factor D Fab by BiaCore

Immobilization—

Human factor D (Advanced Research Inc. 0.1 mg/ml) was directlyimmobilized onto the CM5 chip (BiaCore) using amine-coupling method. Theprocedure is briefly described as following: (1) Constant flow (PBS) isat 5 μl/min. (2) Injection of 35 μl EDC/NHS (1:1). (3) Injection of 35μl of human factor D in acetate buffer, pH 4.5. (4) Block the activatedgroup by injection of 35 μl etholamine. (5) Clean-up the surface by 5 μl10 mM Glycine pH 1.5. The ligand (human factor D) immobilization levelis about 1,000 RU. Test run using α-human factor D (huDi, 40 μl, 31.5μg/ml) yielded a relative response around 900 RU.

Kinetic Analysis—

All anti-human factor D Fabs were diluted in PBS buffer. Each sample wasprepared in a series of concentrations: 12.5 nM, 25 nM, 50 nM, 75 nM,100 nM, 125 nM, and 150 nM, with 40 μl injection pulse at highacquisition rate. Regeneration was accomplished by applying 5 μl pulseof 10 mM Glycine at pH 1.5. Kinetic parameters were obtained by fittingFabs binding traces to 1:1 binding model under pseudo-first orderkinetic using BIAvaluation version 3.0. The results are presented inTable 2 below. All data are obtained by global fitting routine.

TABLE 4 BIAcore Results Fab Clone ka (M−1s−1) kd (s−1) Kd (M)Anti-factor D Fab 315 7.1 × 10⁵ 2.7 × 10⁻⁴ 3.7 × 10⁻¹⁰ Anti-factor D Fab416 8.2 × 10⁵ 1.8 × 10⁻⁴ 3.3 × 10⁻¹⁰ Anti-factor D Fab 345 6.8 × 10⁵ 3.5× 10⁻⁴ 5.1 × 10⁻¹⁰ Anti-factor D Fab 250 5.7 × 10⁵ 1.9 × 10⁻⁴ 3.3 ×10⁻¹⁰ Anti-factor D Fab 56 3.6 × 10⁵ 9.8 × 10⁻⁴ 2.7 × 10⁻⁹  chimeric 4.4× 10⁵ 1.2 × 10⁻³ 2.7 × 10⁻⁹  Anti-factor D Fab 111 3.3 × 10⁵ 3.7 × 10⁻⁴1.14 × 10⁻⁹  

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A murine antibody comprising: (a) a heavy chain variable domainsequence of SEQ ID NO: 1; (b) a light chain variable domain sequence ofSEQ ID NO: 2; or (c) a heavy chain variable domain sequence of SEQ IDNO: 1 and a light chain variable domain sequence of SEQ ID NO:
 2. 2.-3.(canceled)
 4. An antibody comprising SEQ ID NO: 13, 14, 15, 16, 17, and18.
 5. A chimeric antibody comprising SEQ ID NO: 1 and SEQ ID NO:
 2. 6.A variable domain of a humanized antibody comprising a heavy chainvariable domain amino acid sequence selected from the group consistingof: SEQ ID NO: 6; SEQ ID NO: 8; SEQ ID NO: 10; and SEQ ID NO:
 12. 7. Avariable domain of a humanized antibody comprising a light chainvariable domain amino acid sequence selected from the group consistingof SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO: 9; and SEQ ID NO:
 11. 8. Ahumanized anti-Factor D antibody comprising: (a) the variable domainsequence of SEQ ID NO: 5 and the variable domain sequence in SEQ ID NO:6; (b) the variable domain sequence of SEQ ID NO: 7 and the variabledomain sequence in SEQ ID NO: 8; (c) the variable domain sequence of SEQID NO: 9 and the variable domain sequence in SEQ ID NO: 10; or (d) thevariable domain sequence of SEQ ID NO: 11 and the variable domainsequence in SEQ ID NO:
 12. 9.-11. (canceled)
 12. A humanized anti-FactorD antibody having a variable light chain comprising a CDR-L1 having thesequence of SEQ ID NO: 16; a CDR-L2 having the sequence of SEQ ID NO:17, 21, or 24, and a CDR-L3 having the sequence of SEQ ID NO: 18, 19, or22.
 13. A humanized anti-Factor D antibody having a variable heavy chaincomprising a CDR-H1 having the sequence of SEQ ID NO: 13 or 25; a CDR-H2having the sequence of SEQ ID NO: 14; and a CDR-H3 having the sequenceof SEQ ID NO: 15 or
 20. 14. A polypeptide comprising the following aminoacid sequence: QX₁QLVQSGX₂E LKKPGASVKV SCKASGYTFT SYGMNWVX₃QA PGQGLEWMGWINTYTGETTYADDFKGRFVF SLDTSVSTAY LQISSLKAED TAX₄YYCX₅REG GVNNWGQGTL VTVSS(SEQ ID NO: 27), wherein X₁ is I or V; X₂ is P or S; X₃ is K or R; X₄ isT or V; and X₅ is E or A.
 15. A polypeptide comprising the followingamino acid sequence:DIQX₆TQSPSSLSX₇SVGDRVTITCITSTDIDDDMNWYQQKPGKX₈PKLLIX₉DGNTLRPGVPSRFSX₁₀GSGX₁₁DFTLTISSLQPEDVATYYCLQSDSLPYTFGQ GTKLEIK (SEQ ID NO: 26),wherein X₆ is V or M; X₇ is M or A; X₈ is P or V; X₉ is S or Y; X₁₀ is Sor G; and X₁₁ is A or T.
 16. A polypeptide comprising the amino acidsequence of any one of SEQ ID NOS: 5-12.
 17. (canceled)
 18. A humanizedantibody comprising the polypeptide of claim
 14. 19. The humanizedantibody of claim 18 further comprising: (a) a polypeptide comprisingthe following amino acid sequence:DIQX₆TQSPSSLSX₇SVGDRVTITCITSTDIDDDMNWYQQKPGKX₈PKLLIX₉DGNTLRPGVPSRFSX₁₀SGSGX₁₁DFTLTISSLQPEDVATYYCLQSDSLPYTFGQ GTKLEIK (SEQ ID NO: 26),wherein X₆ is V or M; X₇ is M or A; X₈ is P or V; X₉ is S or Y; X₁₀ is Sor G; and X₁₁ is A or T; or (b) a polypeptide comprising the followingamino acid sequence:DIQX₆TQSPSSLSX₇SVGDRVTITCITSTDIDDDMNWYQQKPGKX₈PKLLIX₉DGNTLRPGVPSRFSX₁₀SGSGX₁₁DFTLTISSLQPEDVATYYCLQSDSLPYTFGQ GTKLEIK (SEQ ID NO: 26),wherein X₆ is V or M; X₇ is M or A; X₈ is P or V; X₉ is S or Y; X₁₀ is Sor G; X₁₁ is A or T; and the amino acid at position 104 of SEQ ID NO: 26is a valine or a leucine.
 20. An antibody fragment of claim
 13. 21. Anisolated nucleic acid comprising the sequence of SEQ ID NO: 3 or
 4. 22.(canceled)
 23. An isolated nucleic acid encoding an antibody of claim13.
 24. An isolated nucleic acid encoding the polypeptide of claim 16.25. A vector comprising the nucleic acid of claim
 23. 26. A vectorcomprising the nucleic acid of claim
 24. 27. A cell line comprising thevector of claim
 25. 28. A composition comprising the antibody of claim13.
 29. A method to treat a complement mediated disorder comprisingadministering the composition of claim
 28. 30. The method according toclaim 29, wherein the disorder is an ocular disease such as age-relatedmacular degeneration or diabetic retinopathy.
 31. A variable domain ofclaim 7 wherein amino acid at position 104 of SEQ ID NO: 7 is a valineor a leucine.
 32. A humanized anti-Factor D antibody of claim 8comprising the variable domain sequence of SEQ ID NO: 7 wherein theamino acid at position 104 of SEQ ID NO: 7 is a valine or a leucine andthe variable domain sequence of SEQ ID NO:
 8. 33. A polypeptide of claim15 wherein the amino acid at position 104 of SEQ ID NO: 26 is a valineor a leucine.
 34. A polypeptide of claim 16 wherein the amino acid atposition 104 of SEQ ID NO: 7 is a valine or a leucine.
 35. A method ofmaking a humanized anti-Factor D antibody or fragment thereof of claim13.
 36. An antibody which competes with murine antibody 166-32 and/orhumanized anti-Factor D antibody clone #56, #111, #250 or #416 and/or anantibody comprising variable domain or HVR sequences of humanizedanti-Factor D antibody clone #56, #111, #250 or #416.
 37. An antibodywhich binds to the same epitope as murine antibody 166-32 and/orhumanized anti-Factor D antibody clone #56, #111, #250 or #416 and/or anantibody comprising variable domain or HVR sequences of humanizedanti-Factor D antibody clone #56, #111, #250 or #416.